Uses of an Immunomodulatory Protein (GMI) from Ganoderma Microsporum

ABSTRACT

The invention provides a method for inhibiting EGF receptor activity comprising contacting an EGF receptor with an immunomodulatory protein (GMI) from  Ganoderma microsporum , or a recombinant thereof. Also provided is a method for treating invasion and metastasis of cancer cells, comprising administering an effective amount of an immunomodulatory protein (GMI) from  Ganoderma microsporum , or a recombinant thereof, to a subject in need of such treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and is a continuation-in-partapplication of pending U.S. patent application Ser. No. 13/890,042,filed May 8, 2013, which is a continuation-in-part application andclaims priority to U.S. patent application Ser. No. 12/826,230, filedJun. 29, 2010 (now patented U.S. Pat. No. 8,476,238 issued Jul. 2,2013). The entirety of the above-mentioned patent applications arehereby incorporated by reference herein and made a part ofspecification.

FIELD OF THE INVENTION

The present invention relates to the new uses of an immunomodulatoryprotein (GMI) from Ganoderma microsporum, a combination therapy of GMIand an anti-cancer agent and a pharmaceutical composition comprising GMIand an anti-cancer agent. In particular, GMI can be used to inhibit EGFreceptor activity, treat invasion and metastasis of cancer cells,inhibit drug-resistance cancer calls and reduce the amount of theanti-cancer drug, or inhibition of drug-resistant cancer cells.

BACKGROUND OF THE INVENTION

Neovascularization is critical for the growth for tumors and isimportant in a variety of angiogenic diseases, such as diabeticretinopathy, arthritis, psoriasis and haemangiomas. More than 70% ofcancer patients die from metastatic dissemination of the initial tumor.Tumor neovascularization is the crucial process for survival of aprimary tumor and for metastatic dissemination. Angiostatic steroids andheparin with anti-angiogenic agents such as protamin have been used astherapies to suppress tumor growth. These therapeutic approaches haveserious limitations, because when the dose of heparin exceeds an optimumlevel for inhibition of angiogenesis, both tumor growth and angiogenesisare stimulated. Also, high doses of cortisone that are required forantiangiogenesis leads to immunosuppression. Acquisition of anangiogenic phenotype marked a transition from hyperplasia to neoplasia.

Growth factors are substances that induce cell proliferation, typicallyby binding to specific receptors on cell surfaces. One such growthfactor is epidermal growth factor (EGF). EGF induces proliferation of avariety of cells in vivo, and is required for the growth of mostcultured cells. EGF is a single-chain polypeptide having a molecularweight of 6 Kd (53 amino acid residues) and three internal disulfidebonds. These three well characterized internal disulfide bonds of theepidermal growth factor peptide define three “loops,” the A, B and Cloops. Generally, the A loop is characterized between amino acidresidues 1-19, the B loop is characterized between residues 20-31, andthe C loop is characterized between residues 34-43. EGF is also known tobe a powerful stimulator of cell proliferation. In particular, EGF hasbeen shown to stimulate the growth of epithelial cell tissue in avariety of preparations. Epithelial growth factor receptor (EGFR) playsan important role in epithelial biology and in many human malignancies.EGFR is related to the viral oncogeny, v-erb B, and is overexpressed inmany human tumors, including brain, bladder, breast, and squamous cellcarcinomas of the head, neck and lung. Thus, EGF-R “activation” is animportant regulatory event in stimulating the division of many normalcells as well as in the aberrant growth of some tumor cells. CompleteEGF peptides, and antibodies which mimic their action, have been used insuch diverse processes as screening for tumoricidal activity andpromotion of wound healing. EGFR is a member of the receptor familycomprising four, highly homologous proteins, HER2, HER3, and HER4 aswell as EGFR. Those proteins in this family consist of an extracellulardomain, a transmembrane domain, and an intracellular tyrosine kinasedomain. Binding of the ligand such as epithelial growth factor (EGF)activates the intracellular tyrosine kinase domain to induceautophosphorylation of the receptor, which initiates the signalingcascade involved in cell proliferation and survival. EGFR is one of themost suitable targets in cancer therapy.

Lung cancer is the most common malignancy among men and women, andremains the leading cause of cancer-related deaths. Non-small lungcarcinoma (NSCLC) accounts for approximately 75-85% of lung cancers.Conventional lung cancer treatments generally show poor clinicalresponse, thus it is of utmost importance to develop novel treatmentstrategies directed against metastasis. EGFR overexpression occurs in40-80% of NSCLCs. The EGFR pathway contributes to the pathogenesis andprogression of human carcinoma, including cell proliferation, apoptosis,angiogenesis and metastatic spread. Notably, EGFR-directed tyrosinekinase inhibitors (TKIs) such as gefitinib (Iressa, ZD1839), lapatinib(Tykerb, GW572016) and erlotinib (Tarceva, OSI-774) have differentsensitivities based on the specific subtypes of NSCLC patients. Inaddition, gefitinib has been used as a single agent in NSCLC with modestefficacy. However, patients respond differently to this agent, and veryrecently these responses have been correlated with the presence ofactivating mutations in the tyrosine kinase domain of EGFR. EGFinteracts with EGFR, leading to receptor dimerization, activation of itskinase activity and autophosphorylation of EGFR on tyrosine residues.EGF is also associated with the growth and invasion of various malignanttumors via different pathways. Several studies have shown that EGF isfrequently elevated in lung cancer, and up-regulation of EGF has beenshown to be related to disease progression and poor prognosis (Gorgouliset al., 1992, Anticancer Res, 12, 1183-1187). This suggests that EGFplays a major role in lung tumorigenesis. Therefore, the EGF/EGFRinteraction may be important for the development of lung cancer.

Herbal therapies have increasingly been considered viable alternativetreatments for cancers. Lingzhi (a species of Basidiomycetes) is anherbal mushroom, used in traditional Chinese medicine for at least 2,000years. Many therapeutic effects have been reported of Lingzhi species,such as immunomodulatory, anti-tumor, hepato-protective, antioxidant,and cholesterol-lowering effects (Jinn et al., 2006, Biosci BiotechnolBiochem, 70, 2627-2634). All of these therapeutic effects are attributedto triterpenoids, polyssacharides, and glycoproteins (Boh et al., 2007,Biotechnol Annu Rev, 13, 265-301; Jinn et al., 2006, Biosci BiotechnolBiochem, 70, 2627-2634). A new glycoprotein class in Lingzhi namedfungal immunomodulatory proteins (FIPs) was recently identified. So far,at least 4 FIPs have been isolated and purified from Ganoderma lucidum,LZ-8, (G. lucidum), including FIP-fve (Flammulina veltipes), FIP-vvo(Volvariella volvacea), FIP-gts (Ganoderma tsugae), and FIP-gja(Ganoderma sinensis) (Hsu et al., 1997, Biochem J, 323 (Pt 2), 557-565;Ko et al., 1995, Eur J Biochem, 228, 244-249; Xuanwei et al., 2008,Planta Med, 74, 197-200). According to a previous study, FIP-gts from G.tsugae, a popular chemopreventive mushroom in Asia, has anti-cancerfunction and is involved in the regulation of hTERT/telomeraseexpression (Liao et al., 2006, Mol Carcinog, 45, 220-229). In addition,FIP-gts inhibits the growth of A549 cancer cells, leading to cell cyclearrest, consequently inducing premature cellular senescence in lungcancer cells. Moreover, FIP-gts results in significant inhibition oftumor growth in athymic nude mice implanted with A549 cells (Liao etal., 2008, Food Chem Toxicol, 46, 1851-1859). US 20100009915 provides amethod for suppressing proliferation of a cancer cell and a method forsuppressing a tumor cell mobility, comprising providing to the tumorcell a purified polypeptide of a fungal immunomodulatory protein, LZ-8.

U.S. Pat. No. 7,601,808 discloses an immunomodulatory protein (GMI)cloned from Ganoderma microsporum and this protein has immunomodulatorefficiency. However, since GMI is a newly found immunomodulatory proteinand its anti-cancer effects have not been investigated, there is still aneed in the art to investigate its anticancer applications.

SUMMARY OF THE INVENTION

One object of the invention is to provide a method for inhibiting EGFreceptor activity comprising contacting an EGF receptor with animmunomodulatory protein (GMI) from Ganoderma microsporum, or arecombinant thereof.

Another object of the invention is to provide a method for treatinginvasion and metastasis of cancer cells, comprising administering aneffective amount of an immunomodulatory protein (GMI) from Ganodermamicrosporum, or a recombinant thereof, to a subject in need of suchtreatment.

A further object of the invention is to provide a pharmaceuticalcomposition, comprising GMI or a recombinant thereof and an-anti canceragent.

Another object of the invention is to provide a method for treatinginvasion and metastasis of a breast cancer, comprising administering aneffective amount of an immunomodulatory protein (GMI) from Ganodermamicrosporum, or a recombinant thereof, to a subject in need of suchtreatment.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the inhibition of GMI and AG1478 in cancer cell migrationin A549 cells. A549 cells (1.2×10⁵ cells/24 well) were treated withincreasing doses of GMI (2, 4, 8 μg/m1) for 24 h. Wound-healing assaywas carried out to evaluate the inhibitory effects of (A) GMI or (B)AG1478 on A549 cell migration. Confluent monolayers of A549 cells werescarred, and repair was monitored microscopically after 24 h oftreatment with (A) GMI or (B) AG1478. The cells migrating into the woundarea were counted on the basis of the dashed line as time zero.

FIG. 2 shows the inhibition of GMI in EGF-induced invasion in A549cells. A549 cells (2×10⁴ cells/well) were seeded onto the upper chamberof membrane and treated with different concentrations of GMI (4, 8, 16μg/ml) for 2 hours. The bottom chamber was filled with DMEM supplementedwith EGF 10 ng/ml or 100 ng/ml. After about 24 h, the invasive A549cells passed through the membrane and were quantified by counting thecells that migrated onto the membrane. Cells were fixed, stained, andcounted as described in the text. The data represent mean±SD.

FIG. 3 shows the effects of GMI on signal transduction pathways in A549cells. A549 cells (5×10⁵ cells/60 mm) were pretreated with variousconcentrations of GMI (4, 16 pg/ml) or AG1478 (EGFR inhibitors) for 8hours prior to incubation for 10 minutes with EGF (as indicated). Celllysates were subject to immunoblotting with phospho-specific EGFR(Y1068) (A and B), anti-phospho-AKT (A), anti-AKT (A),anti-phospho-GSK3β (A) or anti-phospho-STAT3 antibodies (B). Proteinloading was determined by Western blotting against β-actin (A and B).

FIG. 4 shows the effect of GMI, PI3K inhibitor and EGFR inhibitor onEGF-induced invasion of A549 cells. EGF (10 ng/ml) was applied to thelower chamber as a chemoattractive agent. Serum-starved cells (2×10⁴cells/well) were seeded onto the upper chamber consisting of 8-μmpore-size filters coated with Matrigel basement membrane matrix and thenincubated with 8 μg/ml GMI, 50 μM Ly294002 (PI3K inhibitor) or 2 μMAG1478 (EGFR inhibitor) for 24 h. Cells that invaded the lower surfaceof the membranes were counted under a light microscope. The data arepresented as mean±SD.

FIG. 5 shows the inhibition of GMI in EGF-induced activation of Cdc42.A549 cells were seeded onto 100 mm plates and cultured to around 80%confluence. The cells were then treated with various doses of GMI (4, 16μg/ml). GMI inhibits Cdc42 activation but has little effect on Rac1.A549 cells were pretreated with various concentrations of GMI for 24 hbefore being stimulated with 10 ng/mL EGF for 3 min. After that, cellswere washed with cold PBS and lysated on the dish in RIPA buffer. ActiveGTP-bound Rac1 or Cdc42 was pulled down using the GST-PBD fusion proteinof PAK1 immobilized on glutathione beads and active Rac1 and Cdc42 weredetected with anti-Rac1 and anti-Cdc42 antibodies.

FIG. 6 shows the effects of GMI on EGF-induced F-actin/G-actin ratio andfilopodia formation. A549 cells (1×10⁴ cells/well) were seeded onto 24wells with coverslip. A549 cells were pretreated with variousconcentrations of GMI (2, 4, 8 μg/ml) for 1 h before being stimulatedwith 10 ng/mL EGF for 23 h. A549 cells treated with or without EGF werestained with Texas Red®-X phalloidin and Alexa Fluor 488 DNase Iconjugate to detect F-actin (red) and G-actin (green). F-actin labelingwith Texas Red®-X phalloidin revealed that A549 cells exhibited numerousfilopodia, whereas GMI-treated cells exhibited fewer filopodia fibers.

FIG. 7 shows the effect of cisplain and GMI co-treatment on CaLu-1 cellsviability.

FIG. 8 shows the cell viability (% of the control) of A549, A549/D16 andA549/V16.

FIGS. 9 (A)-(I) shows the dose response curve of the GMI to various celllines of leukemia (A), non-small cell lung cancer (B), colon cancer (C),CNS cancer (D), melanoma (E), ovarian cancer (F), renal cancer (G),prostate cancer (H) and breast cancer (I)

FIG. 10 shows the one dose Mean Graphs of the GMI to various cell lines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that an immunomodulatoryprotein (GMI) from Ganoderma microsporum is effective in inhibiting EGFreceptor activity and treating invasion and metastasis of cancer cells.Particularly, the GMI inhibits EGF-induced invasive growth of cancercells, including migration and invasion, through blocking of theEGF/phosphoEGFR-PI3K/Akt pathway. GMI induces a dose-dependent decreasein invasion with increasing concentrations of GMI. The invention alsofound significant changes in actin following GMI repression ofEGF-induced activation of EGF/phosphoEGFR-PI3K/Akt pathway, so GMI canbe used as a strong actin deploymerizer in tumor cells. These findingssuggest that GMI has considerable potential for cancer chemoprevention,including inhibiting drug-resistance cancer calls. In particular, GMI incombination with an anti-cancer drug(s) provides advantageous effect(preferably synergistic effect, reduction of amount of the anti-cancerdrug, or inhibition of drug-resistant cancer cells) in the treatmentand/or prevention of cancer or in the inhibition of metastasis orinvasion.

In one aspect, the invention provides a method for inhibiting EGFreceptor activity comprising contacting an EGF receptor with animmunomodulatory protein (GMI) from Ganoderma microsporum, or arecombinant thereof.

In another aspect, the present invention provides a method forinhibiting and/or treating invasion and metastasis of cancer, comprisingadministering an effective amount of an immunomodulatory protein (GMI)from Ganoderma microsporum, or a recombinant thereof, to a subject inneed of such treatment. In one embodiment, the treatment of invasion andmetastasis is through blocking of the EGF/phosphoEGFR-PI3K/Akt pathway.Particularly, the invention provides a method for treating invasion andmetastasis of a breast cancer, comprising administering an effectiveamount of an immunomodulatory protein (GMI) from Ganoderma microsporum,or a recombinant thereof, to a subject in need of such treatment.Preferably, the GMI has the amino acid sequences of:(1)-Leu-Ala-Trp-Asn-Val-Lys-(LAWNVK; SEQ ID NO:1) and(2)-Asp-Leu-Gly-Val-Arg-Pro-Ser-Tyr-Ala-Val-(DLGVRPSYAV; SEQ ID NO:2) orthe amino acid sequence ofMSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN (SEQ ID NO:3).

In a further aspect, the present invention provides a method forinhibiting and/or treating cancer and/or invasion and metastasis ofcancer, comprising administering GMI or a recombination thereof and ananti-cancer agent simultaneously, sequentially or separately. In anotherfurther aspect, the present invention provides a pharmaceuticalcomposition, comprising GMI or a recombinant thereof and an-anti canceragent.

In another aspect, the present invention provides a pharmaceuticalcomposition, comprising GMI or a recombinant thereof and an-anti canceragent. Preferably, the composition exhibits a synergist effect intreating and/or preventing cancer.

Preferably, the cancer is mediated by EGFR receptor.

According to the invention, the immunomodulatory protein (GMI) is fromGanoderma microsporum or a recombinant thereof. More preferably, the GMIhas the amino acid sequences: (1)-Leu-Ala-Trp-Asn-Val-Lys-(LAWNVK; SEQID NO:1) and (2)-Asp-Leu-Gly-Val-Arg-Pro-Ser-Tyr-Ala-Val-(DLGVRPSYAV;SEQ ID NO:2) or the amino acid sequence of:MSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN (SEQ ID NO:3).

According to the invention, the terms “treatment,” “treating” and thelike are used herein to generally mean obtaining a desiredpharmacologic, physiologic or cosmetic effect. The effect may beprophylactic in terms of completely or partially preventing a condition,appearance, disease or symptom and/or may be therapeutic in terms of apartial or complete cure for a condition and/or adverse effectattributable to a condition or disease. “Treatment” as used hereincovers any treatment of a condition, disease or undesirable appearancein a mammal, particularly a human, and includes: (a) preventing thedisease (e.g. cancer), condition (pain) or appearance (e.g. visibletumors) from occurring in a subject which may be predisposed to it buthas not yet been observed or diagnosed as having it; (b) inhibiting thedisease, condition or appearance, i.e., causing regression of conditionor appearance; (c) relieving the disease, condition or appearance, i.e.,causing regression of a condition or appearance.

The term “inhibit” as used herein with reference to cancer invasion andmetastasis refers to any reduction in cancer invasion and metastasis byGMI or pharmaceutical composition of the invention.

The term “effective amount” is the quantity of compound which achieves abeneficial clinical outcome when the compound is administered to asubject. For example, when a compound of the invention is administeredto a subject with a cancer, a “beneficial clinical outcome” includesreduction in tumor mass, reduction in metastasis, reduction in theseverity of the symptoms associated with the cancer and/or increase inthe longevity of the subject.

According to the invention, the term “metastasis” or “invasion” refersto the ability of a cell to migrate through a physiological barrier orto protease components of an extracellular matrix. Preferredphysiological barriers include basement membranes and otherextracellular matrices which are well known in the art. Cell invasion iscorrelated to the secretion or excretion of proteolytic enzymes from acell. Preferred proteolytic enzymes include MMPs.

In one embodiment, the invasion and metastasis of cancer cells isepidermal growth factor mediated migration and invasion of cancer cells.Epidermal growth factor (EGF) is a small molecule which exhibitshomology with regions of the TGF-alpha molecule. It is produced bymacrophages and epidermal cells with the keratinocyte and fibroblast astargets. Its primary role is to stimulate keratinocytes to migrateacross a wound's provisional matrix and induce epidermal regeneration.EGF, like all growth factors, binds to specific high-affinity,low-capacity receptors on the surface of responsive cells. Intrinsic tothe EGF receptor is tyrosine kinase activity, which is activated inresponse to EGF binding. The kinase domain of the EGF receptorphosphorylates the EGF receptor itself (autophosphorylation) as well asother proteins, in signal transduction cascades, that are associatedwith the receptor following activation. The activation of EGFR is highlyinvolved in the processes of tumor proliferation and progression,including cell proliferation, inhibition of apoptosis, angiogenesis andmetastasis. EGFR shows relatively high expression in epithelial cancersand the expression correlates with tumor progression, and therefore itis one of the most suitable targets in cancer therapy.

Preferably, the cancer is lung cancer (more preferably, non-small lungcarcinoma, NSCLC), squamous cell carcinomas of the lung, head and neck,breast cancer, ovarian cancer, prostate cancer, gastric carcinoma,cervical cancer, esophageal carcinoma, bladder cancer, brain cancer,liver cancer, or colon cancer.

GMI can be administered to a patient either alone or in pharmaceuticalcompositions where it is mixed with suitable carriers and excipients.GMI can be administered parenterally, such as by intravenous injectionor infusion, intraperitoneal injection, subcutaneous injection, orintramuscular injection. GMI can be administered orally or rectallythrough appropriate formulation with carriers and excipients to formtablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like. GMI can be administered topically, such as by skin patch.GMI can be formulated into topical creams, skin or mucosal patch,liquids or gels suitable to topical application to skin or mucosalmembrane surfaces. GMI can be administered by inhaler to the respiratorytract for local or systemic treatment of cancers. In one embodiment, theamount of GMI for administration may ranges from 250 μg to 500 μg for ahuman with 60 kg.

The dosage of GMI suitable for use according to the present inventioncan be determined by those skilled in the art on the basis of thedisclosure herein. The medicament will contain an effective dosage(depending upon the route of administration and pharmacokinetics of theactive agent) of GMI and suitable pharmaceutical carriers and excipientswhich are suitable for the particular route of administration of theformulation (i.e., oral, parenteral, topical or by inhalation). GMI ismixed into the pharmaceutical formulation by means of mixing,dissolving, granulating, dragee-making, emulsifying, encapsulating,entrapping or lyophilizing processes. The pharmaceutical formulationsfor parenteral administration include aqueous solutions of the inventivepolypeptide in water-soluble form. Additionally, suspensions of theinventive polypeptide may be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension mayoptionally contain stabilizers or agents to increase the solubility ofthe complex or combination to allow for more concentrated solutions.

In one embodiment, GMI can be used in combination with radiotherapy andchemotherapy.

In another embodiment, the GMI or a recombinant thereof can be combinedwith an anti-cancer agent for combination therapy in cancer and/orcancer invasion and metastasis. GMI a recombinant thereof also can becombined with an anti-cancer agent as a pharmaceutical composition. Thatis, the invention provides a pharmaceutical composition comprising GMIor a recombinant thereof and an anti-cancer agent and the compositioncan treat and/or prevent cancer or treat and/or prevent cancermetastasis or invasion. Preferably, the cancer is lung cancer (morepreferably, non-small lung carcinoma, NSCLC), anal cancer, squamous cellcarcinomas of the lung, head and neck, breast cancer, ovarian cancer,prostate cancer, gastric carcinoma, cervical cancer, esophagealcarcinoma, bladder cancer, brain cancer, liver cancer, or colon cancer.Particularly, the composition preferably exhibits a synergistic efficacyor reduced amount of the anti-cancer drug or increased inhibition todrug-resistant cancer cells. According to one embodiment of theinvention, the anti-cancer agent include, but are not limited to: amitotic inhibitor (such as taxanes (preferably paclitaxel, docetaxel),vinca alkaloids (preferably, vinblastine, vincristine, vindesine andvinorelbine) and vepesid; an anthracycline antibiotic (such asdoxorubicin, daunorubicin, daunorubicin, epirubicin, idarubicin,valrubicin and mitoxantrone); a nucleoside analog (such as gemcitabine);an EGFR inhibitor (such as gefitinib and erlotinib); an folateantimetabolite (such as trimethoprim, pyrimethamine and pemetrexed);cisplatin and carboplatin. According to one embodiment of the invention,the the anti-cancer agent include, but are not limited to:20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, pro staticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cisplatin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypernycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfo sine; edrecolomab; eflomithineklerriene; emitefur;epirubicin; epristeride; erlotinib; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine;fluorodaunoruriicin hydrochloride; forfenimex; formestane; fostriecin;fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine;ganirelix; gefitinib; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immuno stimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;lapatinib; leinamycin; lenograstim; lentinan sulfate; leptolstatin;letrozole; leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; lo soxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor- saporin; mitoxantrone; mofarotene; molgramo stim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipid A,niyobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;

ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronicacid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfo sfamide; perillyl alcohol; phenazinomycin;phenylacetate; pho sphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;

plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2, proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; rarnosetran; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonerrnin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

According to the invention, the anti-cancer agent can be a therapeuticantibody, including but not limiting to HERCEPTIN® (Trastuzumab)(Genentech, Calif.), which is a humanized anti-HER2 monoclonal antibodyfor the treatment of patients with metastatic breast cancer; REOPRO®(abciximab) (Centocor), which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland), which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX®, which is amurine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2, which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225, which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN®, which is a humanizedanti-.alpha.V.beta.3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti CD52IgG1 antibody (Leukosite); Smart M195, which is a humanized anti-CD33IgG antibody (Protein Design Lab/Kanebo); RITUXAN®, which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE®, which is a humanized anti-CD22 IgG antibody (Immunomedics);LYMPHOCIDE® Y-90 (Immunomedic s); Lympho scan (Tc-99m-labeled;radioimaging; Immunomedics); Nuvion (against CD3; Protein Design Labs);CM3, which is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114,which is a primatied anti-CD80 antibody (IDEC Pharm/Mitsubishi);ZEVALIN®, which is a radiolabelled murine anti-CD20 antibody(IDEC/Schering AG); IDEC-131, which is a humanized anti-CD4OL antibody(IDEC/Eisai); IDEC-151, which is a primatized anti-CD4 antibody (IDEC);IDEC-152, which is a primatized anti-CD23 antibody (IDEC/Seikagaku);SMART anti-CD3, which is a humanized anti-CD3 IgG (Protein Design Lab);5G1.1, which is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7, which is a humanized anti-TNF-alpha antibody(CAT/BASF); CDP870, which is a humanized anti-TNF-alpha Fab fragment(Celltech); IDEC-151, which is a primatized anti-CD4 IgG1 antibody (IDECPharm/SmithKline Beecham); MDX-CD4, which is a human anti-CD4 IgGantibody (Medarex(Eisai/Genmab); CD20-sreptdavidin+biotin-yttrium 90(NeoRx); CDP571, which is a humanized anti-TNF-alpha IgG4 antibody(Celltech); LDP-02, which is a humanized anti-alpha-4-beta-7 antibody(LeukoSite/Genentech); OrthoClone OKT4A, which is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA®, which is a humanized anti-CD40LIgG antibody (Biogen); ANTEGREN®, which is a humanized anti-VLA-4 IgGantibody (Elan); and CAT-152, which is a human anti-TGF-beta 2 antibody(Cambridge Ab Tech).

In a further embodiment, the anti-cancer agent can be selected from thegroup consisting of cisplatin, gefitinib, lapatinib and erlotinib.

The present invention evaluates the inhibitory effect of GMI on invasionand metastasis of cancer cells (especially, EGF-induced invasion andmetastasis of cancer cells) and investigates the molecular mechanisminvolved. The present invention demonstrates that EGF treatment leads toPI3K/Akt increase, which results in upregulation of Racl/Cdc42 activityand assembly of cell-cell contacts, as well as enhanced invasion bytumor cells. GMI inhibits EGF-induced A549 cell migration and invasioninvolving both PI3K/Akt and Stat3 pathways. Furthermore, GMI can inhibitthe TNF-alpha induced MMP9 protease activity.

Without further elaboration, it is believed that one skilled in the artcan utilize the present invention to its fullest extent on the basis ofthe preceding description. The following examples are, therefore, to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way.

EXAMPLE Example 1 GMI Inhibits EGF-induced Migration and Invasion inA549 Cells

Recombinant human EGF was purchased from Peprotech Inc. AG1478 wasobtained from Calbiochem. Antibodies used for Western blotting againstPhospho-specific EGFR Y1068 (ab40815) and cdc42 (ab41429) were obtainedfrom Abcam. Anti-phospho STAT3 Y705 (#9131), anti-phospho GSK-3β (#9336)and anti-phospho Akt Ser473 (#9271) were purchased from Cell Signaling.Other antibodies included monoclonal anti-β actin antibody (Sigma) andRac1 antibody (Upstate). GMI manufactured by Mycomagic BiotechnologyCo., Ltd., was used in the examples and it was generated and amelioratedfrom Ganoderma microsporum (see, U.S. Pat. No. 7,601,808, which isincorporated with in its entity for reference). The GMI used in theexample and the subsequent examples has the amino acid sequence as shownin SEQ ID NO: 3. The human lung carcinoma cell line, A549 [American TypeCulture Collection (ATCC); CCL-185], was obtained from ATCC. The cellswere grown in DMEM (Life Technologies) supplemented with 10%heat-inactivated fetal bovine serum (FBS; Life Technologies), as well aspenicillin and streptomycin (100 mg/mL each), at 37° C. in a humidifiedatmosphere of 5% CO₂.

Expression of EGFR in small cell lung cancer cells has been previouslydemonstrated (Jaramillo et al., 2008, Cancer Biol Ther, 7, 557-568). Tounderstand the inhibitory effect of GMI on EGF-promoted migration inA549 cells, the effects of GMI on EGF-induced cell motility andwound-healing assay were investigated.

Wound-Healing Assay

On wound-healing assay, the cancer cells were cultured onto 24-wellplates and grown in medium containing 10% FBS to nearly confluent cellmonolayer. A plastic pipette tip was used to draw a linear “wound” inthe cell monolayer of each well. The monolayer was then washed twicewith PBS to remove debris or detached cells, and GMI was added atdifferent concentrations (0, 2, 4, and 8 mg/mL). PBS was added to thecontrol well as the solvent control. After 24 h of incubation, the cellswere washed twice with PBS, fixed with 95% alcohol, and stained with 10%Giemsa solution (Merck). The cells were photographed under a lightmicroscope (magnification, ×200). The experiments were performed intriplicate. After incubation with 2-8 μg/ml of GMI for 24 h, the cellsthat migrated to the denuded zone were photographed. The resultsdemonstrated that GMI dose-dependently suppresses A549 cell migration tothe denuded zone (FIG. 1A). In contrast, the cells treated only with EGFshowed acceleration of wound closure after treatment for 24 h (FIG. 1A).Cells co-treated with EGF and GMI demonstrated markedly decreased woundclosure activity (FIG. 1A). On the other hand, AG1478, a positivecontrol for EGF, was able to inhibit EGFR phosphorylation. AG1478inhibition of EGF-induced migration was similar to that of GMI (FIG.1B). Cell invasion assay

Cell invasion assays were performed using modified Boyden chambers 6.5mm in diameter, with 10 mm thick porous (8 μm) polycarbonate membraneseparating the two chambers (Transwell; Costar, Cambridge, MA). Themembrane of the upper chamber was coated with Matrigel (0.3 mg/mL; BDBiosciences Discovery Labware) for 3 hours. Condition medium wasprepared from A549 cells which were pretreated with or without GMI,LY294002 (50 μM), or AG1478 (2 μM) for 8 h. To the medium of the bottomchamber was added 10% FBS-DMEM containing EGF (10 ng/ml) or conditionmedium. Cells were trypsinized, centrifuged, and resuspended at 4×10⁵cells/mL in 0.5% FBS-DMEM. After 24 h incubation, the cells on the upperwell and the membranes coated with Matrigel were fixed with methanol,and stained with 20% Giemsa solution (Merck). The cells that wereattached to the lower surface of the polycarbonate filter were countedunder a light microscope (magnification, X 100). The experiments wereperformed in triplicate.

The invasive ability of tumor cells is one of the importantcharacteristics of metastasis. Boyden chamber assay was modified toquantify the invasive potential of A549 cells. The results showed thatGMI induced a dose-dependent decrease in invasion with increasingconcentrations of GMI (FIG. 2A). At 4 μg/ml the invasion was reduced to22% (invaded cell number decreased from 1895±37 to 419±6) and at 16μg/ml the invasion was reduced to less than 9% (invaded cell numberdecreased from 1895±37 to 61±3). Subsequently, GMI induced adose-dependent decrease in EGF-promoted invasion (FIG. 2A). At 4 μg/mlthe invasion was reduced to 52% (invaded cell number decreased from2515±212 to 1306±30) and at 16 μg/ml the invasion was reduced to lessthan 5% (invaded cell number decreased from 2515±212 to 121±39). Theresults demonstrated that GMI significantly inhibits EGF-promotedinvasion of A549 cells.

Example 2 GMI Inhibits EGF-induced Phosphorylation of EGFR, Akt andStat3

Several studies have indicated that the transcription factors p38 MAPK,Stat3 and Akt are involved in cell metastasis activity in different celltypes (Broadbelt et al., 2009, Am J Physiol Renal Physiol, 297,F114-124; Lee et al., 2008, Toxicol Appl Pharmacol, 226, 178-191; Shihet al., 2009, Food Chem Toxicol, 47, 1985-1995). Akt acts downstream ofPI3K. It is a multifunctional regulator of cell survival, growth andinvasion, and is phosphorylated within 10 min of EGF stimulation. Theseobservations suggest that activation of Akt by EGF plays a role in theinvasive activities of A549 cells.

Western Blotting

Total cell lysates were prepared in RIPA buffer (100 μl in 60mm indish). Fifty μg of lysate were loaded onto an 8% polyacrylamide gel andanalyzed by SDS gel electrophoresis. After transfer to PVDF (Amersham),blots were blocked with 5% skim milk in TBS (10 mM Tris-CL pH7.5, 150 mMNaC1) containing 0.2% Tween-20. Blots were probed with antibodies (attheir recommended dilutions) in 5% skim milk in TBS overnight at 4° C.Following detection with the appropriate horseradish-peroxidaseconjugated secondary antibody (Cell Signaling), blots were developed byenhanced chemiluminescence according to the manufacturer's directions(Perkin Elmer Life Sciences). All experiments were performed induplicate.

Actin Staining

Cells were washed twice with PBS and fixed in a 3.7%paraformaldehyde-PBS solution for 10 min at room temperature. After twoadditional washes with PBS, cells were permeabilized with a solution of0.1% Triton X-100 in PBS for 3 to 5 min and washed again with PBS. TexasRed-X phalloidin (2 units/mL) and Alexa Fluor 488 DNase I conjugate (9μg/mL) were used to localize filamentous actin (F-actin) and G-actin.Fluorescent dyes were diluted with blocking solution (1% bovine serumalbumin and 0.025% saponin in PBS) and added to coverslips for 60 min atroom temperature. After three washes with PBS, coverslips were mountedon a microscope slide with Prolong Gold antifade reagent with DAPI (LifeTechnologies). F-actin cytoskeleton imaging was performed with aconfocal laser scanning microscope (ZEISS LSM510 BETA) at ×400magnification.

To assess whether GMI mediates and/or inhibits phosphorylation of Aktand Stat3, the invention investigated the effect of GMI on thephosphorylation status of Akt and Stat3 in A549 cells treated withvarious concentrations of GMI for 8h. FIGS. 3 show that GMIsignificantly inhibits EGF-induced activation of EGFR and Akt, whereasit has little effect on Stat3.

Example 3 GMI Inhibits Cdc42 Activity and Microfilament Depolymerizationin A549 Cells

EGF induces cell proliferation and migration mainly through activationof its cell surface receptor EGFR. Rac1 activating signaling pathwaysare located downstream of EGFR (Binker et al., 2009, Biochem Biophys ResCommun, 379, 445-450).

Rac1 and Cdc42 Activity Assay

Active Rac1 and Cdc42 were determined by pull-down assay (Binker et al.,2009, Biochem Biophys Res Commun, 379, 445-450). Serum-starved A549cells were or were not stimulated for 3 min with EGF and then collectedin 800 μl of ice-cold lysis-buffer. Lysates were centrifuged to removecellular debris. From each supernatant, 5 μl were taken out to measureprotein content using Protein Assay Kit (Bio-Rad, Hercules, Calif.).Twenty μl were removed to determine total Rac1 in total lysate, and theremaining volume was used for the pull-down assay. Lysates containingequal amounts of proteins were then mixed with 15 μg ofGST-PAK-PBD-beads (Pierce). Samples for total Rac1 and Cdc42 in totallysate and the pelleted beads were diluted in Laemmli sample-buffer andboiled. The proteins were separated using SDS-PAGE (12% gel). Aftertransfer to nitrocellulose membranes (Bio-Rad), blots were blocked withbovine serum albumin, followed by incubation with Rac1 antibodyovernight. Binding of the antibody was visualized usingperoxidase-coupled anti-mouse antibody and chemiluminescence method(Perkin Elmer Life Sciences). Equal loading was verified by reprobingmembranes corresponding to total lysate with anti-β-actin antibody (notshown).

To determine the relationship between the anti-invading effect of GMIand Rac1/Cdc42 signaling pathway, the effect of GMI on Rac 1 activitywas evaluated. As shown in FIG. 5 (Lanes 1 and 2), Rac1 and Cdc42activities are induced by 10 ng/ml EGF for 3 min. In contrast, GMIreduced EGF-promoted Cdc42 activity in a dose-dependent manner, withlittle reduction in Rac1 activity in A549 cells (FIG. 5, lane 2 to lane4). However, Rac1 activity correlated directly with cell mobility.

Given that reorganization of the actin cytoskeleton is a criticaldeterminant of cellular invasion, the invention next analyzed theG-actin and F-actin cytoskeletal architecture in GMI-treated cells withEGF (Denys et al., 2008, Cancer Lett, 266, 263-274). EGF plays animportant role in the progression of breast carcinomas, and aspreviously shown (Lu et al., 2003, Cancer Cell, 4, 499-515). EGF at 10ng/ml over 72 h can induce EMT in A431 cells. In this study, as shown inFIG. 6, EGF induced F-actin fibers (red) and increased G-actin fibers(green). Lamellipodia formations were observed in A549 cells with EGFfor 24 h. GMI abrogated elongation and polymerization of F-actin andG-actin in A549 cells treated with EGF or GMI alone. Focal contact sizewas also reduced by GMI in a dose-dependent manner and with combinedtreatment.

Example 4 Effect of Cisplatin and GMI Co-Treatment on CaLu-1 or A549Cell Viability

CaLu-1 cells were co-treated with various concentrations of cisplatin(0, 2.5, 5, 10, 20 μM) and GMI from Ganoderma microsporum(0, 4, 8 and 16μg/mL) for 48 h followed by MTT assay to estimate cell viability. Thesynergistic effect was determined after co-treated Cisplatin and GMI.

MTT assay was used to determine the effect of GMI and cisplatin on theproliferation of Calu-1 cells. In metabolically active cells, MTT(Thiazolyl Blue Tetrazolium Bromide) (Sigma) was reduced bydehydrogenase enzyme into an formazan product. Absorbance was measureddirectly at 570 nm from 96-well assay plates after adding 100 μl DMSO.The quantity of formazan was considered to be directly proportional tothe number of viable cells in the culture.

Briefly, the cells (5×10³) were incubated on 96-well plates containing200 μl of growth medium. After 24 h incubation, the medium was carefullyremoved and 100 μl of fresh medium containing various concentrations ofGMI and cisplatin were added to the wells. The cells were treated withGMI and cisplatin continuously for 48 h with 0, 4, 8, 16 μg/ml for GMIand with 0, 2.5, 5 and 10 μM for cisplatin. At the end of this process,100 μl/well of 0.5 mg/ml MTT solution was added and wells were incubatedfor 3 h, 37° C., in a humidified incubator. The supernatant was aspiredand 100 μl DMSO was added therein to solve the formazan. The absorbancewas analyzed on a VERSAmax microplate reader at 570 nm. Absorbancevalues were presented as the mean±SE of 3 replicates for each treatment.Cells in controls and compound controls were included. Absorbance ofuntreated cells was considered 100%. As shown in FIG. 7, the combinationof GMI and cisplatin exhibits a synergistic effect and the amount ofcisplatin can be largely reduced when it is combinatorially used withGMI. For example, 2.5 μM cisplatin in combination with 16 μg/ml GMI canachieve about 50% cell viability, whereas cisplatin alone needs about 10μM to achieve the same viability.

Example 4 Effect of GMI on Cell Viability of Drug Resistant A549 CellSublines

A549 cell line and resistant A549 cell sublines (A549/D16, A549/D32 andA549/V16) (5000 cells/well of 96-well plate) were treated with increaseddoses concentrations (μM) of GMI from Ganoderma microsporum, docetaxel,vincristine and doxorubicin for 48 h, respectively. Cell viability wasmeasured by MTT assay and the results are presented as the calculatedcell growth inhibitory ratio. Experiments were repeated three times. Theresults of the drug sensivity of A549 cell line and the drug resistantsublines are shown in Table 1.

TABLE 1 Drug sensitivity of parental A549 cell line and the drugresistant sublines. IC₅₀ ± SD* (nmol/L) Drug A549 A549/D16 A549/D32A549/V16 Docetaxel  6.4 ± 0.1 (1.0)  730.0 ± 56.6 (114.1) 2035.0 ± 954.6(318.0)  780.0 ± 51.3 (121.9) Vincristine  14.9 ± 0.1 (1.0)  770.0 ±56.6 (51.9)  730.0 ± 28.3 (49.2)  760.0 ± 92.4 (51.2) Doxorubicin 222.7± 23.2 (1.0) 3423.9 ± 152.3 (15.4) 7694.4 ± 148.0 (34.6) 1531.4 ± 80.8(6.9) GMI (μg/ml) 15.73 ± 0.42  11.37 ± 0.55 not detected  13.10 ± 0.17

The data of cell viability (% of the control) of A549, A549/D16 andA549/V16 are shown in FIG. 8.

Example 5 NCI60 in vitro Screening Assay for Inhibition of Cancer Cellsby GMI

The US National Cancer Institute (NCI) 60 anticancer drug screen (NCI60)tumour cell lines are screened for the activity of GMI and the materialsand methods of NCE 60 can refer to Shoemaker, Robert H., Nature Reviews,vol. 6, Oct. 2006, pp. 813-823. The design of the NCI60 in vitroscreening model required concentration-response testing for eachcompound in each member of the cell line panels, so as to assess therelative potency of test compounds across the cell lines. The “MeanGraph” provides a compact way of presenting the profile of relativesensitivity and resistance of all the cell lines at three levels ofeffect: 50% growth inhibition (GI50), total growth inhibition (TGI) and50% lethal concentration (LC50), and the “COMPARE” algorithm provides anautomated way of comparing these profiles with all or parts of thescreening database.

The NCI60 in vitro screening model for testing the cancer cellinhibition of the GMI to a number of cancer cell lines was conducted byNational Institutes of Health (NIH), Maryland, U.S.A. The dose responsecurve of the GMI to various cell lines of leukemia, non-small cell lungcancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renalcancer, prostate cancer and breast cancer were shown in FIG. 9 (A)-(I).The GI50, TGI and LC50 of the GMI to various cell lines are shown inTable 2. The one dose Mean Graphs of the GMI to various cell lines areshown in Table 3 and FIG. 10. The Mean Graphs of the GMI to various celllines are shown in Table 4.

TABLE 2 Log10 Concentration Mean Optical Densities Percent GrowthPanel/Cell Line Time Zero Ctrl −1.9 −0.9 0.1 1.1 2.1 −1.9 −0.9 0.1 1.12.1 GI50 TGI LC50 Leukemia CCRF-CEM 0.402 1.731 1.784 1.741 1.686 1.1780.645 104 101 97 58 18 2.02E1 >1.25E2 >1.25E2 HL-60(TB) 1.031 3.1253.129 3.110 3.228 3.224 2.952 100 99 105 105 92 >1.25E2 >1.25E2 >1.25E2K-562 0.366 2.530 2.449 2.489 2.555 2.463 2.319 96 98 101 9790 >1.25E2 >1.25E2 >1.25E2 MOLT-4 1.017 2.973 2.937 2.911 3.040 2.2391.034 95 97 103 62 1 1.99E1 >1.25E2 >1.25E2 RPMI-8226 1.220 3.162 3.2523.209 3.259 3.155 2.610 105 102 105 100 72 >1.25E2 >1.25E2 >1.25E2 SR0.675 2.729 2.691 2.637 2.648 1.026 0.703 98 96 96 17 14.79E0 >1.25E2 >1.25E2 Non-Small Cell Lung Cancer A549/ATCC 0.376 1.9601.992 1.948 1.871 1.183 0.496 102 99 94 51 8 1.31E1 >1.25E2 >1.25E2HOP-62 0.394 1.026 1.050 1.040 1.036 0.853 0.260 104 102 102 73 −342.03E1 5.99E1 >1.25E2 NCI-H226 1.075 2.571 2.567 2.562 2.587 2.055 1.421100 99 101 65 23 2.90E1 >1.25E2 >1.25E2 NCI-H23 0.853 2.326 2.318 2.3482.413 2.170 1.822 99 101 106 89 66 >1.25E2 >1.25E2 >1.25E2 NCI-H322M0.800 1.833 1.855 1.829 2.044 1.846 1.403 102 100 120 10158 >1.25E2 >1.25E2 >1.25E2 NCI-H460 0.220 2.425 2.487 2.506 2.416 2.2310.958 103 104 100 91 33 6.47E1 >1.25E2 >1.25E2 NCI-H522 1.032 2.3562.291 2.353 2.425 2.325 1.731 95 100 105 98 53 >1.25E2 1.25E2 >1.25E2Colon Cancer COLO 205 0.616 2.564 2.524 2.520 2.556 2.459 2.468 95 98100 95 95 >1.25E2 >1.25E2 >1.25E2 HCC-2998 1.089 2.934 2.982 2.920 3.0292.798 2.718 103 99 105 93 88 >1.25E2 >1.25E2 >1.25E2 HCT-116 0.235 1.7731.740 1.844 1.795 1.825 1.837 98 105 101 103 104 >1.25E2 >1.25E2 >1.25E2HCT-15 0.325 2.285 2.222 2.236 2.175 1.340 0.224 97 97 94 52 −31 1.31E15.26E1 >1.25E2 HT29 0.259 1.590 1.584 1.579 1.560 1.014 0.552 100 99 9857 22 1.95E1 >1.25E2 >1.25E2 KM12 0.498 2.376 2.379 2.293 2.447 2.1691.311 100 96 104 89 43 8.92E1 >1.25E2 >1.25E2 SW-620 0.357 2.480 2.5232.474 2.521 2.373 1.482 102 100 102 95 53 >1.25E2 >1.25E2 >1.25E2 CNSCancer SF-268 0.551 1.840 1.816 1.856 1.944 1.354 0.597 95 101 108 52 42.02E1 >1.25E2 >1.25E2 SF-295 1.121 3.050 3.117 3.042 3.070 2.914 1.551103 100 101 93 22 5.07E1 >1.25E2 >1.25E2 SF-539 0.846 2.544 2.688 2.7302.578 2.207 0.531 108 111 102 80 −37 2.26E1 6.02E1 >1.25E2 SNB-19 1.2492.501 2.471 2.472 2.441 2.078 0.525 98 98 95 66 −58 1.69E1 4.27E1 1.08E2SNB-75 0.760 1.624 1.583 1.633 1.599 0.801 0.247 95 101 97 5 −68 4.04E01.45E1 7.14E1 Melanoma MALME-3M 0.807 1.662 1.663 1.675 1.818 1.7791.437 100 102 118 114 74 >1.25E2 >1.25E2 >1.25E2 M14 0.449 1.646 1.6181.601 1.722 1.357 0.465 98 96 106 76 1 2.78E1 >1.25E2 >1.25E2 MDA-MB-4350.563 2.371 2.389 2.335 2.203 2.096 0.621 101 96 91 85 33.34E1 >1.25E2 >1.25E2 SK-MEL-28 0.493 1.644 1.622 1.678 1.754 1.6161.321 98 103 110 98 72 >1.25E2 >1.25E2 >1.25E2 SK-MEL-5 1.002 3.3443.367 3.369 3.405 3.200 0.175 101 101 103 94 −83 2.22E1 4.26E1 8.17E1UACC-257 0.860 1.895 1.931 1.887 1.879 1.760 1.397 103 99 98 3752 >1.25E2 >1.25E2 >1.25E2 UACC-62 0.834 2.627 2.712 2.648 2.615 2.7322.054 105 101 99 106 68 >1.25E2 >1.25E2 >1.25E2 Ovarian Cancer IGROV10.563 1.912 1.946 1.891 2.143 1.397 0.968 103 98 117 62 302.94E1 >1.25E2 >1.25E2 OVCAR-3 0.478 1.515 1.508 1.531 1.600 1.359 0.74899 102 108 85 26 4.90E1 >1.25E2 >1.25E2 OVCAR-4 0.716 1.325 1.347 1.3351.336 1.365 1.095 103 102 102 107 62 >1.25E2 >1.25E2 >1.25E2 OVCAR-50.545 1.558 1.568 1.563 1.539 1.449 1.215 101 100 98 8966 >1.25E2 >1.25E2 >1.25E2 OVCAR-8 0.550 2.115 2.089 2.030 2.097 1.9731.032 98 95 99 91 31 6.00E1 >1.25E2 >1.25E2 NCI/ADR-RES 0.625 1.9601.990 1.958 2.029 1.704 0.878 102 100 105 81 19 3.94E1 >1.25E2 >1.25E2SK-OV-3 0.579 1.202 1.222 1.222 1.248 1.164 0.920 103 103 107 9455 >1.25E2 >1.25E2 >1.25E2 Renal Cancer 786-0 0.659 2.385 2.380 2.3622.573 1.919 0.733 100 99 111 73 4 2.70E1 >1.25E2 >1.25E2 A498 1.3112.092 2.149 2.108 2.199 2.153 0.846 107 102 114 108 −35 3.17E17.07E1 >1.25E2 ACHN 0.374 1.661 1.775 1.653 1.694 0.834 0.218 109 99 10336 −42 7.65E0 3.62E1 >1.25E2 CAKI-1 0.823 2.961 2.962 2.960 2.914 1.8210.706 100 100 98 47 −14 1.08E1 7.29E1 >1.25E2 RXF 393 0.796 1.291 1.2961.297 1.294 1.172 0.353 101 101 101 76 −56 1.97E1 4.72E1 1.13E2 SN12C0.735 2.614 2.655 2.690 2.591 2.251 1.300 102 104 99 81 305.05E1 >1.25E2 >1.25E2 TK-10 0.846 1.794 1.754 1.774 1.848 0.236 −0.02796 98 106 −72 −100 2.57E0 4.91E0 9.38E0 UO-31 0.543 1.798 1.760 1.8282.034 1.015 0.123 97 102 119 38 −77 8.79E0 2.65E1 7.22E1 Prostate CancerPC-3 0.470 1.458 1.495 1.482 1.435 1.196 0.112 104 102 98 73 −76 1.79E13.87E1 8.34E1 DU-145 0.404 1.531 1.542 1.525 1.630 1.209 0.507 101 99109 71 9 2.76E1 >1.25E2 >1.25E2 Breast Cancer MCF7 0.170 1.184 1.1861.223 1.117 0.667 0.400 100 104 93 49 23 1.15E1 >1.25E2 >1.25E2MDA-MB-231/ 0.651 1.484 1.511 1.447 1.427 0.991 0.346 103 96 93 41 −478.34E0 3.65E1 >1.25E2 ATCC HS 578T 1.254 2.273 2.317 2.332 2.357 2.2341.907 104 106 108 96 64 >1.25E2 >1.25E2 >1.25E2 BT-549 0.991 1.973 2.0081.957 1.972 1.244 0.262 104 98 100 26 −74 5.89E0 2.27E1 7.24E1 T-47D0.577 1.413 1.420 1.380 1.312 0.934 0.687 101 96 88 43 138.63E0 >1.25E2 >1.25E2 MDA-MB-468 0.802 1.607 1.642 1.608 1.726 0.7800.611 104 100 115 −3 −24 4.45E0 1.18E1 >1.25E2

TABLE 3 Panel/Cell Line Growth Percent Leukemia HL-60(TB) 116.48 K-562125.37 MOLT-4 44.75 RPMI-8226 96.01 SR 32.47 Non-Small Cell Lung CancerA549/ATCC 44.29 HOP-62 53.89 HOP-92 22.29 NCI-H226 83.77 NCI-H23 91.82NCI-H322M 91.20 NCI-H460 86.38 NCI-H522 125.90 Colon Cancer COLO 205105.56 HCC-2998 132.04 HCT-116 101.77 HCT-15 33.64 HT29 28.36 KM12 62.96SW-620 87.57 CNS Cancer SF-268 39.72 SF-295 70.58 SF-539 48.19 SNB-1958.70 SNB-75 −45.73 U251 69.04 Melanoma LOX IMVI 67.70 MALME-3M 114.17M14 94.87 MDA-MB-435 79.29 SK-MEL-2 105.92 SK-MEL-28 106.96 SK-MEL-583.26 UACC-257 89.25 UACC-62 115.96 Ovarian Cancer IGROV1 30.07 OVCAR-394.57 OVCAR-4 98.08 OVCAR-5 107.85 OVCAR-8 76.68 SK-OV-3 89.18 RenalCancer 786-0 74.75 A498 98.16 ACHN −33.94 CAKI-1 96.04 RXF 393 38.93SN12C 74.30 TK-10 −92.37 UO-31 14.01 Prostate Cancer PC-3 79.73 DU-14539.22 Breast Cancer MCF7 42.68 MDA-MB-231/ATCC 23.69 BT-549 −74.30 T-47D28.25 MDA-MB-468 50.90 Mean 64.66 Delta 157.03 Range 224.41

What is claimed is:
 1. A method for treating invasion and metastasis ofa breast cancer, comprising administering an effective amount of animmunomodulatory protein (GMI) from Ganoderma microsporum, or arecombinant thereof, to a subject in need of such treatment, wherein theGMI has the amino acid sequences of:(1)-Leu-Ala-Trp-Asn-Val-Lys-(LAWNVK; SEQ ID NO:1) and(2)-Asp-Leu-Gly-Val-Arg-Pro-Ser-Tyr-Ala-Val-(DLGVRPSYAV; SEQ ID NO:2) orthe amino acid sequence ofMSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN (SEQ ID NO:3). 2.The method according to claim 1, wherein GMI is administeredparenterally.
 3. The method according to claim 1, wherein GMI isadministered orally or rectally.
 4. The method according to claim 1,wherein GMI is used in cancer chemoprevention.
 5. The method accordingto claim 1, wherein GMI is administered in combination with radiotherapyand chemotherapy.
 6. The method according to claim 1, wherein the methodfurther comprise administrating an anti-cancer drug selected from amitotic inhibitor, an anthracycline antibiotic, a nucleoside analog, anEGFR inhibitor, or an folate antimetabolite.
 7. The method according toclaim 6, wherein the mitotic inhibitor is paclitaxel, docetaxelvinblastine, vincristine, vindesine, vinorelbine or vepesid; theanthracycline antibiotic is doxorubicin, daunorubicin, daunorubicin,epirubicin, idarubicin, valrubicin or mitoxantrone; the nucleosideanalog is gemcitabine), the EGFR inhibitor is gefitinib or erlotinib);and the folate antimetabolite is trimethoprim, pyrimethamine orpemetrexed.
 8. The method of claim 6, wherein GMI or a recombinationthereof and the anti-cancer agent is administered simultaneously,sequentially or separately.